Process for producing a large amount of isoamylase

ABSTRACT

According to the present invention, a strain of Aerobacter aerogenes is engaged in a preliminary culture upon a streak culture and then one platinum loop of the resulting seed culture is inoculated into a liquid medium sterilized as usual, which medium contains ammonium salt as a nitrogen source and liquefied starch as a carbon source, and is cultured under the condition of pH 5-8 to produce a large amount of isoamylase.

United States Patent Kazuo Masuda Okayam-shi, Okayama;

Masakazu Mitsuhashi, Okayama-shi, Okayama; Mamoru Hirao, Setocho, Akaiwa-gun, Okayama; Yoshinori Sato, Okayama-shi, Okayama; Kaname Sugimoto, Okayama-shi, Okayama, all of [72] Inventors Japan 21 1 Appl. No. 733,325 [22] Filed May 31, 1968 [45] Patented Nov. 23, 1971 i [73] Assignee l-layashibara Co.

Okayama-shi, Okayama, Japan [32] Priority June 1, 1967 [33] Japan [31] 42/34468 [54] PROCESS FOR PRODUCING A LARGE AMOUNT OF ISOAMYLASE 2 Claims, No Drawings [52] U.S.Cl 195/66 R,

Wallentels et al., Biochemical and Biophysical Research Communications Vol. 22 No.3 pg. 254- 261 (1966).

Primary Examiner-Lionel M. Shapiro Attorneys-Alvin Browdy and Sheridan Neimark ABSTRACT: According to the present invention, a strain of Aerobacter aerogenes is engaged in a preliminary culture upon a streak culture and then one platinum loop of the resulting seed culture is inoculated into a liquid medium sterilized as usual, which medium contains ammonium salt as a nitrogen source and liquefied starch as a carbon source, and is cultured under the condition of pH 5-8 to produce a large amount of isoamylase.

PROCESS FOR PRODUCING A LARGE AMOUNT OF ISOAMYLASE This invention relates to a process of producing a large amount of isoamylase. More particularly, this invention relates to a method of producing isoamylase by the culture of the organism producing isoamylase upon a culture medium, which consists of ammonium salts as nitrogen source and liquefied starch as carbon source, to produce and accumulate a large amount of isoamylase in the culture medium.

lsoamylase is an enzyme which catalyzes to specifically hydrolyze alpha l.6-linkage at the branched position of starch.

It was reported by Bender et al. in 1961 that isoamylase (they named this as pullulanase could be obtained by the method of the culture of a strain of Aerobacter aerogenes.

Bender et al. reported that an enzyme solution with high activity was produced by the culture of using a culture medium which contains inorganic or organic nitrogen as nitrogen source and the enzyme as an induction enzyme, being especially inducted with maltose, maltotriose and pullulan.

The present inventors concentrated their efforts in paying attention to the substrate specificity of the isoamylase, in the search of an industrial method for producing a large amount of isoamylase. As the result, it has been found that when using liquefied starch as the carbon source and ammonium salts as the nitrogen source a large amount of isoamylase may be produced and accumulated in the culture medium.

An object of the present invention is to provide an isoamylase which has high activity.

A further object of the present invention is to provide a novel process for producing a large amount of isoamylase by the culture of Aerobacter aerogenes.

A still further object of the present invention is to produce an isoamylase with a high degree of activity in a simple and efficient manner in high yield.

The present invention will be more fully described hereunder following some experimental examples.

Bender et al. reported that the culture medium of the composition given in the following tables 1 and 2 Aerobactor inoculated with Aerobactor aerogenes and the strain cultured to produce from to units/ml. of isoamylase (also named pullulanase) produced in the culture. But this method isnt satisfactory for industrial purposes.

Table I Table 2 Na NO: 0.3% Na NO: 0.5%

K,H PO; 0. l% Peptone 0.8%

KCL 0.05% Mg so,-114,o 0.05%

Fe SO-7H2O 0.001% KCl 0.05%

Maltose 0.8% Fe sc -711,0 0.001%

Maltese 0.5%

The determination of enzyme activity shown hereunder was conducted as follows:

A reaction mixture consisting ol'enzyme solution 1 ml. 1% soluble glutinous rice starch solution 5 ml. 0.5M acetate buffer solution I ml.

EXPERIMENT l A streak culture medium composed of Peptone l 0% Yeast extract 0.5% Mg s0,-7H,o 005% KCl 0.05% Fe so,-7H,o 0.001% Maltese l.0%

is inoculated with Aerobacter aerogenes and the strain is cultured for 20 hours (this culture is used in this experiment as a seed culture as well as in the experiments and examples hereafter).

A medium composed of (NH ),SO 0.4% K," P0, 0.7% Kl-LPO; 0.3% Mg S0.7H,0 0.05% KC] 0.05% Fe S0.-7H,O 0.00M Multose. soluble starch 0r liquefied starch 1.4%

which was poured into a 500 ml. conical flask and sterilized as usual was inoculated with one platinum loop of the resulting seed culture and the charge was then cultured at 30 C. for 40 hours. Thereafter pH, absorbency and enzyme activity of the resulting solution obtained were determined. The results are tabulated in the following table.

TABLE 3 Transition of lsoamylase Activity with Carbon Sources Carbohydrate used pH Abscrbency Enzyme activity as substrate (unit/ml.)

Soluble starch 7.80 0.500 35 Maltese 6.90 0.570 39 Liquified starch 7.70 0.520 I38 Notes: The liquefied starch refers to a solution wherein a commercially available liquei'ying enzyme (alpha-amylase) was added to a potato starch emulsion to be treated and the pH of it was adjusted to about 6 and continuously Iiquified at 88 As the results from table 3 show it has been found that the use of liquefied starch in the culture medium is obviously successful for producing an amylase.

EXPERIMENT 2 The following culture media A, B and C, each of which have different nitrogen sources, were cultured in the same manner as described in experiment 1. However, the pl-l of culture medium C was adjusted to 3 with alkali.

Culture medium A in the method described in experiment 1, after changing the kinds of nitrogen source and the mount used in culture medium 1.4 percent of liquified starch is used as carbon source), the materials to be treated were cultured at 30 C. for 25 hours.

TABLE 5 Influence of nitrogen sources and its concentration on isoamylase activity Amount Enzyme used, activity percent pH Absorbency (unit/ml.)

0. 172 7. 40 0. 015 0. 344 7. 45 0. 017 0 3. 0 6. 45 0. 325 0. 485 7. 20 0. 015 0 0. 070 6. 98 O. 120 2 0. 230 6. 82 0. 400 5 0. 460 6. 75 0. 420 20 0. 920 6. 48 0. 530 20 0. 40 6. 0 0. 210 30 Ammonium ac 0. 60 8. 8 0.222 80 Indicates when periods of the culture are 72 hours.

According to the results shown in tables 4 and 5, it has been found that an enzyme with high activity may be produced when using ammonium salts as the nitrogen source in the culture medium. The ammonium salts that can be used for the production and the accumulation of isoamylase include inorganic ammonium slats such as ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonium chloride and organic ammonium salts such as ammonium acetate.

ln order to carry out the present invention, it is desirable to employ a liquefied starch with liquefaction ratio from 5 to percent as the carbon source and the amount used in the culture medium is preferably from 1 to 3 percent. These matters may be determined from the results of experiment 4 and 5 60 described hereafter.

EXPERIMENT 4 0.48 percent of NH NO instead of the (NHJ SQ, in experiment 1 was used as the nitrogen source in the culture medium and L4 percent of liquefied starches which have difierent liquefaction ratios were used as the carbon source.

The culture was conducted by the same method as described in experiment 1. The temperature for the culture was 30 C. and the period was for 55 hours. The results are as shown in table 6.

TABLE 6 Relations between D.E. (dextrose equivalent) of liquified starch and the isoamylase activity The results from table 6 show that it is preferred to use liquefied starch with from about 5 to about 10 DE. as the carbon source in the culture medium to produce isoamylase.

Bender et al. also reported that when using maltose as the carbon source, the isoamylase activity increased until 0.8 percent of the maltose was used. However, it may be found from the results in experiment 5 that when using liquefied starch as the carbon source, the isoamylase activity increases until 1.4 percent of the liquefied starch is used and it is suitable to use from I to 3 percent of the liquefied starch.

EXPERIMENT 5 Following the method of experiment 1, the results as shown in table 7 were obtained. Liquefied starch was used as the car bon source.

TABLE 7 Relations between the amount added of liquefied starch and activity of isoamylase obtained Moreover, the amount of ammonium salts used as the nitrogen source in the culture medium with the aforesaid liquefied starch can be above 0.2 percent as shown in table 5.

In order to obtain an enzyme with high activity, the pH of the culture fluid should be kept above 5 during the culture.

Accordingly, when operating according to the present method, the pH of the culture fluid during the culture is kept above 5 by increasing the concentration of phosphate in the culture field, which phosphate acts as buffer, or, in addition, by the addition of alkaline materials, as, for example, NaOH, 29. L .99&C9? SEQ-1. IJRF RW fl flt hc beginning period of the culture or during the culture.

For example, following the method of experiment l, the addition of 0.5 percent by weight of calcium carbonate to the culture fluid at the beginning period of the culture and the addition of sodium hydroxide to the culture fluid during the culture were compared with respect to their enzyme activities after 48 hours from the start of the culture. The results are as following table 8.

Relations between an adjustment of pH with alkali and enzyme activity EXAMPLE 1 A streak culture medium composed of Pcptone 1.0% Yeast extract 0.5%

M gSO H O 0.05% KCI 0.05% FeSO -H,O 0.001% Maltose 1.0%

is inoculated with Aerobacter aerogenes and the strain is engaged in a preliminary culture at 30 C. for hours. Then one platinum loop of the slope culture was inoculated into a liquid medium, sterilized as usual, which medium was composed of 1. 0.4% K2HPOI 0.7% J t 0.3% s H i 0.05% KCl 0.05% Fe.so.-7H.o 0.001% liquefied starch V and was cultured under the conditions of pH 5-7 at C. for 48 hours. The activity of the resulting isoamylase obtained was 138 units/ml.

EXAMPLE 2 Following the method of example 1, sodium hydroxide was added to the liquid medium during the culture and cultured under a pH of 5-7 for 48 hours. The activity of the resulting isoamylase obtained was units/ml.

The liquefied starch that can be used in this invention may be either acid-liquefied starch or enzyme liquefied starch which may be prepared by known methods in the field of starch conversion. When used in the claims, the term liquefied starch" accordingly is meant to refer to either acidliquefied starch or enzyme-liquefied starch. For example, an acid-liquefied starch may be prepared by pouring an aqueous starch solution containing 0.3 percent of oxalic acid into boiling water and heating for 10 minutes at 2 atmospheric pressure.

What is claimed is:

1. In a process for the production of isoamylase by the culture of a strain of Aeorbacter aeragenes to produce and accumulate isoamylase in a culture medium, the improvement which comprises adding liquefied starch of DE about 5-10 as a carbon source and ammonium salt as a nitrogen source to the culture medium.

2. In a process for the production of isoamylase by the culture of a strain of Aerabacter aerogenes to produce and accumulate isoamylase in a culture medium, the improvement which comprises adding 1-30 percent liquefied starch of DE. 5-10 as carbon source and more than 0.2 percent of a nitrogen source selected from the group consisting of ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonium chloride and ammonium acetate, to the culture medium and maintaining a pH of the culture medium above 5 during the culture.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 622 6 Dated Nov. 23, 1971 Inventofls) Kazuo MASUDA et a1.

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, line 11,9 (Table l) "MgSO H O" should read --MgSO 7H 0-- Column 3, line 53, delete "slats" and insert --salts-- Column 5, line 12, delete "MgSO H O" and insert --MgSO 7H O- Signed and sealed this 13th day of June 1972.

(SEAL) Attest:

EDWARD M.FLETCHER, JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents ORM PO-105OU0'59' USCOMM-DC 60376-PB9 a LI 5 GOVERNMENT FRINYING OFFICE .99 0-356-334 

2. In a process for the production of isoamylase by the culture of a strain of Aerobacter aerogenes to produce and accumulate isoamylase in a culture medium, the improvement which comprises adding 1-30 percent liquefied starch of D.E. 5-10 as carbon source and more than 0.2 percent of a nitrogen source selected from the group consisting of ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonium chloride and ammonium acetate, to the culture medium and maintaining a pH of the culture medium above 5 during the culture. 